Genetic testing to determine the presence of or a susceptibility to a disease condition offers incredible opportunities for improved medical care, and the potential for such testing increases almost daily as ever increasing numbers of disease-associated genes and/or mutations are identified. One such disease-associated gene is the BRCA1 gene. Mutations in the BRCA1 gene have been shown to be linked to breast and ovarian cancer. Miki et al., xe2x80x9cA Strong Candidate for the Breast Cancer and Ovarian Cancer Susceptibility Gene BRCA1xe2x80x9d, Science 226: 66-71 (1994).
Since the identification of the BRCA1 gene, researchers have tested many individuals using sequencing, single-stranded conformational polymorphism, allele-specific oligonucleotide hybridization and heteroduplex analysis to identify BRCA1 mutations in families with a history of breast and ovarian cancer. Shattuck-Eidens et al., xe2x80x9cA Collaborative Survey of 80 Mutations in the BRCA1 Breast and Ovarian Cancer Susceptibility Genexe2x80x9d, J. Amer. Med. Assoc. 273: 535-541 (1995). Diagnostic screening to identify persons with these mutations, and thus with an apparent genetic predisposition to breast and ovarian cancers offers the opportunity for increased monitoring of high-risk patients which should lead to earlier detection of cancer. Such early detection improves the likelihood of successful treatment and the likelihood of long-term post-detection survival. On the other hand, large scale screening could be prohibitively expensive, absent a easily-performed, low-cost test for mutations in the BRCA1 gene.
It as an object of the present invention to provide a screening methodology which can be used to provide low-cost testing for mutations in the BRCA1 gene.
It is a further object of the present invention to provide reagents, particularly primers and primer cocktails, which can be used in testing for mutations in the BRCA1 gene.
In accordance with the present invention, samples are tested for mutations in the BRCA1 gene using a hierarchical approach. First, each sample is amplified in one or more multiplex PCR amplification reactions. Each multiplex PCR reaction produces a mixture of amplified fragments. The sizes and amounts of these fragments are evaluated and compared to standard values reflecting the sizes and amounts of fragments produced when the same multiplex amplification is performed on the wild-type BRCA1 gene. Differences between the observed fragment sizes and/or amounts and those for the wild-type gene are indicative of a mutation with the BRCA1 gene of the sample.
The next step in the method of the invention is sequencing of one or more regions within the BRCA1 gene. In accordance with the hierarchical method, such sequencing will be performed on samples where no mutation was detected by analysis of the multiplex PCR fragments.